| Background and Objective: The Epstein-Barr virus(EBV)-encoded BARF1 gene is considered as one of the virus oncogene and it frequently expressed in tissues of the EBV related epithelial tumors, indicating that BARF1 gene may play important roles in tumorigenesis. Viral interleukin IL-10(v IL-10) encoded by BCRF1 gene of EBV is highly homologous with human IL-10(h IL-10), primarily mediates immunosuppressive functions and may involve in maintaining immune escape and latent infection. Different variation spectrum was found among nasopharyngeal carcinomas(NPC), EBV-associated gastric carcinomas and healthy donors from Shandong area in our previous studies on polymorphisms of BARF1 and v IL-10 genes. The purpose of this study is to characterize the variations of BARF1 and v IL-10 genes in NPC and healthy donors from Guangzhou area, where NPC is endemic, and to further explore the association of BARF1 and v IL-10 variations with NPC.Methods: PCR and DNA sequencing were used to detect the sequences of the BARF1 and v IL-10 genes in isolates from NPC tissues and throat washing specimens of healthy donors in Guangzhou area. The sequences of BARF1 and v IL-10 genes were compared with the prototype B95-8 strain and classified according to the signature changes and the phylogenetic tree. The distributions of BARF1 and v IL-10 variants were compared between NPC and healthy donors and between Guangzhou and Shandong areas. The genotypes at Bam HI F region were detected by PCR and restricted fragment length polymorphism and the linkages among Bam HI F, BARF1 and v IL-10 variants were also analyzed.Results: ①A total 123 isolates from 50 NPC cases and 73 healthy donors were successfully sequenced in BARF1 gene and three variants were identified. The sequences with an amino acid mutation V29 A were arranged into V29 A group, and the sequences which showed identical amino acid sequence to B95-8 but carried a synonymous mutation, t165545c(aa14), were characterized into B95-8t42 c group,while the sequences with identical nucleotide sequence to B95-8 or those which only carried sporadic mutations were arranged into B95-8P group. B95-8t42 c was more common in NPC cases(76%, 38/50) than healthy donors(50.7%, 37/73)(χ2=7.992, P=0.005). NPC cases in Guangzhou area showed higher frequency of B95-8t42 c but lower frequency of V29A(4%, 2/50) than NPC cases in Shandong area(B95-8t42 c, 26.4%, 24/91; V29 A, 23.1%, 21/91)(χ2=32.856, P<0.0001), and healthy donors in Guangzhou area also showed higher frequency of B95-8t42 c than healthy donors in Shandong area(7.6%, 6/79)(χ2=34.726, P<0.0001).②A total 120 isolates from 50 NPC and 70 healthy donor samples were successfully sequenced in v IL-10 gene. Among them, 42(84.0%) NPC isolates and 60(85.7%) healthy donor isolates with identical amino acid sequences to B95-8 prototype were arranged into one B95-8 group, while the remaining 8(16.0%) NPC isolates and 10(14.3%) healthy donor isolates with amino acid mutations at the signal peptide region were characterized into SPM group. The distribution of the two patterns of v IL-10 gene between NPC and healthy donors was not significantly different(χ2=0.067, P=0.795). The v IL-10 variants in both NPC cases and healthy donors were similarly distributed between Guangdong area and Shandong area(NPC, P=0.073; healthy donors, P=0.691).③The type f showed higher frequency in NPC cases(70.9%, 39/55) than healthy donors(39.4%, 28/71)(χ2=12.329, P=0.0004). By integrating the variations associated with Bam HI F, BARF1 and v IL-10 genes, 12 independent EBV substrains(Bam HI F/BARF1/v IL-10) were identified in isolates which were available of sequences in all three genetic loci. The distributions of the combinational genotypes at three genetic loci were significant different between population groups and between two areas(P<0.0001). The frequency of type f/B95-8t42c/B95-8 in NPC cases from Guangzhou area(62%, 31/50) was significantly higher than that in healthy donors from Guangzhou area(32.8%, 21/64) or NPC cases from Shandong area(1.2%, 1/81). The distribution of BARF1/v IL-10 genotypes in cases with type f EBV was different from that in cases with type F(P<0.0001). The cases with type F corresponded to various BARF1/v IL-10 genotypes, whereas the cases with type f mainly corresponded to B95-8t42c/B95-8 genotype, accounting for 91.2%(52/57) of the cases with type f.Conclusions: ①BARF1 gene shows different variation spectrum between Guangzhou and Shandong areas. The B95-8t42 c variant is the dormant BARF1 variant in Guangzhou area, and it may be associated with NPC especially endemic NPC. ②The variation of v IL-10 gene in Guangdong area is similar with that in Shandong area and B95-8 is the dominant type of v IL-10 in Guangzhou. The v IL-10 variation may have no association with NPC. ③A preferential linkage between Bam HI F and BARF1/v IL-10 polymorphisms exists and type f isolates correlates with the B95-8t42c/B95-8 genotype in endemic NPC isolates. The higher prevalence of type f/ B95-8t42c/B95-8 strains in NPC may also suggest their association with endemic NPC. |
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